MXPA06011171A - Ex-vivo application of solid microparticulate therapeutic agents. - Google Patents

Abstract

The present invention is concerned with a method of preparing and delivering small particles of a pharmaceutically active material to a mammalian subject for treating diseases or disorders. A preferred embodiment entails: (i) the collection of tissue cells from an animal donor, (ii) selective or non-selective growth of these cells in a cell culture medium to which is added solid particles of a therapeutically active compound, mostly free of a drug carrier (about 10% or less, by weight), and having an average particle size of less than about 100 microns, (iii) contacting the cells in the cell culture medium with the solid particles of therapeutically active compound causing the particles to be taken up by the cells into either the intracellular compartment of the cultured cells, attachment of the active compound as particles to the periphery of such cells, or a combination of intracellular uptake and attachment to the cell surface, (iv) optionally, isolation and/or resuspension of the cells prepared in steps i through iii, (v) administering the cells to the mammalian subject. The pharmaceutically active material can be administered intravenously, intramuscularly, subcutaneously, intradermally, intra-articularly, intrathecally, epidurally, intracerebrally, via buccal route, rectally, topically, transdermally, orally, intranasally, via pulmonary route, intraperitoneally, or combinations threof. After administration, the loaded cells transport the pharmaceutical composition as particles.

Description

encode secreted factors that could have broad applications ranging from the treatment of hereditary simple gene deficiencies to acquired disorders of the vasculature or cancer. Myoblasts have recently been transfected via retroviral vectors (Ozawa CR, Springer ML, BIau HM, A novel means of drug delivery: myoblast-mediated gene therapy and retroviral regulatable vectors (A novel means of drug delivery: myoblast-mediated gene therapy and adjustable retroviral vectors.) Annu Rev Pharmacol Toxicol 2000, volume 40, pages 295-317). In the area of pharmaceutical delivery of small molecules, Bender et al. Describe the treatment of HIV-infected monocytes / macrophages with polyhexylcyanoacrylate nanoparticles, either with the nucleoside analogue zalcitabine (2 ', 3'-dideoxycytidine) or saquinavir, a protease inhibitor (Bender et al., Efficiency of Nanoparticles as Carrier System for Antiviral Agents Human Immunodeficiency Virus-lnfected Human Monocytes / Macrophages In Vitro, Antimicrobial Agents and Chemotherapy, (Efficiency of nanoparticles as a carrier system for antiviral agents in human monocytes / macropheres infected with human immunodeficiency virus in vitro, agents antimicrobials and chemotherapy), June 1996, volume 40 (6), p.1467-1471). The polyhexylcyanoacrylate nanoparticles were prepared by emulsion polymerization and tested in-vitro for antiviral activity in primary human monocytes / macrophages. An aqueous solution of saquinavir showed little antiviral activity in macrophages infected with H IV, whereas the nanoparticuide formulation showed significant antiviral activity at one tenth of the solution concentration. At a concentration of 1 00 nM, saquinavir in solution was completely inactive in chronically infected HIV-infected macrophages, but when it was bound to nanoparticles it caused a 35% decrease in viral antigen production. In this study, the drug was trapped in a polymer matrix (polyhexylcyanoacrylate). The preparation of solid, pure drug nanoparticles for delivery to macrophages was not described. The particles were only delivered to in-vitro macrophages and did not contemplate drug delivery when administering cells treated with nanoparticles that were capable of transporting the drug. US Patents Nos. 4,972,465 (Baurain et al.) And 5,100,591 (Leclef et al.), Describe lipid microparticles of nystatin, amphotericin B and other anti-fungal compounds, having potentially intensified focus for macrophages. The present invention overcomes the disadvantages of the prior art by providing pharmaceutical compositions comprising solid pharmaceutical agents in an ex vivo drug delivery method. The advantage to this approach over the use of an attached matrix (ie, direct patient dosing formulation) is that the high drug load is achieved and a high explosion of medication can be delivered. As part of this invention, we describe the ex-vivo application of solid submicron particles that are primarily free of a carrier matrix and consist only of solid medicament with active surface stabilizing ingredients. The solid particulate is contacted with cells capable of surrounding the particles or in which the particles are capable of binding to the outer surfaces of the cells. The different therapeutic areas are appropriate for this ex-vivo technology: bacterial, viral and fungal treatment, neoplasms, lysosomal storage disorders, autoimmune disorders and metabolic disorders. Gene delivery and delivery of antisense oligonucleotides are also described. The ex vivo delivery of nanoparticles, in particular particles of essentially pure active agent, can provide many advantages in several therapeutic areas. For example, in gene therapy, vectors are used normally since they can be either viral or non-viral. For their convenience, viruses offer strong specificity and high transfection efficiency via the natural mechanism of the virus to deliver DNA to the cell. Having expressed the in-vivo gene, fatal, unintended consequences can also be induced, particularly if the unwanted expression of the viral genetic material occurs. On the other hand, non-viral approaches, although less toxic, are relatively inefficient and non-specific. However, nonviral vectors are based on carrier vesicles for the nucleic acid. U.S. Patent Publication no. US 2003/0092069 attempts to remedy the non-specific delivery of genes, in vivo, by describing the delivery of specific site genes via a hollow nanoparticle. More specifically, the publication '069 describes the use of a bio-recognition molecule (protein L of hepatitis B virus) for the delivery of a protein to a hepatocyte. Pharmaceutical agents can be delivered to phagocytize cells in ex vivo culture by adding a solution. However, most of the drug can not be concentrated enough in cells when the influx is based on the molecular diffusion of drug solute through membranes. This inefficient use of medication may require extracellular perfusion of higher drug concentrations, which can produce cytotoxic effects in the culture. A more efficient delivery of the drug can be carried out by leveraging the capacity of phagocytes (for example, macrophages, monocytes, reticulocytes, eosinophils, basophils, neutrophils and dendritic cells, among others) to surround particles. The present invention overcomes the above limitations by delivering a microparticulate of pharmaceutical agent, substantially free of carrier, by cells that are capable of surrounding the microparticulate, or by adsorbing the microparticulate of pharmaceutical agent on the cell surface, and upon which the delivery of The cells charged to the patient is able to reach the target tissues.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for delivering a pharmaceutical composition for targeting cells of a mammalian subject by cellular transport. More specifically, the method of the present invention involves: (1) the collection of tissue cells from a mammalian donor, (2) the selective or non-selective growth of these cells in a cell culture medium to which is added a pharmaceutical composition comprising solid particles of a therapeutically active compound, preferably, almost free of a medicament carrier (about 10% or less, by weight), for the purpose of delivering the active compound to either the intracellular compartment of the cultured cells, binding of the active agent to the periphery of such cells, or a combination of intracellular uptake and binding to the cell surface, (3) optionally, isolation and / or resuspension of the cells prepared in steps 1 and 2, and (4) administration of the cell suspension prepared in steps 1 to 3 in a mammal. After isolation of the mammalian subject, the cells are placed in contact with the pharmaceutical composition comprising particles of pharmaceutical agents. The cells can take the particles through phagocytosis or adsorption of the particle on the surface of the cell, or by specific site delivery (eg, use of a bio-recognition molecule in conjunction with a lipid or other hollow nanoparticle) . Phagocytic cells include, but are not limited to, macrophages, monocytes, granulocytes, neutrophils, basophils, eosinophils, and dendritic cells. Any other type of cell that will surround or adsorb a microparticle of medicament can be used in the present invention. Such cells to be echolated and dosed ex vivo include, but are not limited to, red blood cells, muscle cells, bone and bone marrow cells, vascular cells, organ tissue cells and neuronal cells. In a preferred form of the invention, during contact with the cells, the particles are at a concentration greater than the solubility of thermodynamic saturation, thereby allowing the particles to remain in particulate form during uptake and subsequent delivery to focus somatic tissues by the cells. The loaded cells can be administered by many routes, including intravenous, intramuscular, subcutaneous, intradermal, intra-articular, intrathecal, epidural, intracerebral, intraperitoneal, intra-ophthalmic, retro-orbital and the like. The loaded cells can be infused, either intermittently or continuously, or injected by syringe. In another preferred embodiment, the method comprises the steps of providing a dispersion of the pharmaceutical composition as particles having an average particle size of less than about 100 microns (preferably less than 10 microns) and administering the dispersion directly to the mammalian subject for delivery to target tissues of a portion of the pharmaceutical composition, by reinfused cells capable of reaching the target tissue. The pharmaceutical composition used in these processes can be prepared as small solid particles and can be a therapeutic agent or a diagnostic agent. The therapeutic agents can include any agent used to treat mammalian diseases, such as, but not limited to, agents for the treatment of bacterial, viral and fungal infections, neoplasms, lysosomal storage disorders, autoimmune, inflammatory disorders and metabolic disorders. Therapeutic agents for gene delivery and delivery of antisense oligonucleotides (ATO) are also described. There are numerous advantages of delivering ex-vivo medication upon in vivo administration. For example, by delivering solid drug particles that are associated (for example, surrounded or adsorbed on the cell surface) with cells, the drug particles can be transported to sites of infection. Phagocytosis is carried out by white blood cells, mainly macrophages, neutrophils and eosinophils. Neutrophils predominate early in infection or inflammation, followed by nomadic macrophages that originate from monocytes that leave the blood vasculature and enter infected tissue. Fixed macrophages (histiocytes) abound in the liver, nervous system, lungs, lymph nodes, bone marrow, and various other tissues. The tissues that are most affected by bacterial, viral or fungal pathogens and which are inflamed, can be focused by delivery of cells loaded with medication (granulocytes, for example), having a propensity to be directed to these sites of inflammation by chemotaxis. The pharmaceutical agent is released from these cells in a region of inflammation with high populations of pathogens. Or, the pharmaceutical agent can be an anti-inflammatory drug (steroid, for example), in which case the drug is released in the region where it is most needed therapeutically.

Excessive liver metabolism of medications can be avoided and the cost of therapy can be reduced through this invention. This concept is further extended in this invention to cover the delivery of small solid particles to any depository for phagocytic cells, recognizing that phagocytes, in particular macrophages, are penetrating into almost all connective tissues throughout the body. Macrophages can be divided into normal and inflammatory macrophages. Normal macrophages include those histiocytes that reside in connective tissue, Kupffer cells of the liver, alveolar macrophages within the lung, free and fixed macrophages within the lymph nodes, spleen (free and fixed macrophages), bone marrow (fixed macrophages), serous fluids (pleural and peritoneal macrophages), skin (histiocytes, Langerhan cells) and in other tissues. The population of macrophages in a specific tissue can be maintained by several competent processes: influx of monocyte from the blood, local proliferation from progenitor cells and change. Inflammatory macrophages are present in extracellular fluids.

DETAILED DESCRIPTION OF THE INVENTION The present invention is susceptible to modalities in many different forms. Preferred embodiments of the invention are described with the understanding that the present description is to be considered as exemplifications of the principles of the invention and is not intended to limit the broad aspects of the invention to the illustrated embodiments.

The present invention provides a method of treatment that involves the ex vivo delivery of pharmaceutical agents to a set of cells followed by reinfusion of the cells now treated to the host. This process can be autologous (same donor and recipient) or heterologous (different donor and recipient). The method generally involves: (1) harvesting tissue cells from an animal donor, (2) selective or non-selective growth of these cells in a cell culture medium to which a pharmaceutical composition comprising solid particles of a therapeutically compound is added. active, preferably substantially free of carrier (defined below), for the purpose of either uptake of the active compound into the intracellular compartment of the cultured cells, binding of the active compound to the periphery of such cells, or a combination of intracellular uptake and binding to the cell surface, (3) optionally, isolating and / or resuspending the cells prepared in steps 1 and 2, and (4) administering the cell suspension prepared in steps 1 to 3 to an animal. The following description of the pharmaceutical composition applies to all embodiments of this invention. The pharmaceutical composition may be poorly soluble in water or soluble in water. The pharmaceutical composition can also be a therapeutic agent or a diagnostic agent. The pharmaceutical composition can also be one or more compounds of similar or different therapeutic class. The therapeutic agents can include any compound that is used to treat diseases in which affected tissues can be reached by cells administered that are loaded with a solid particulate form of the compound. Such diseases include, but are not limited to, bacterial, viral and fungal infections, neoplasms, lysosomal storage disorders, inflammatory and autoimmune disorders, and metabolic disorders. Inflammatory and autoimmune disorders may include, but are not limited to, osteoarthritis, rheumatoid arthritis, Crohn's disease, cystitis, ileitis, colitis, lupus, multiple sclerosis, and amyotrophic lateral sclerosis. "Substantially carrier free" refers to a solid particle consisting of substantially pure pharmaceutical agent (approximately 90% or more than 90% by weight), with surfactants or other excipients added to the supersension to either prevent aggregation or modulate the release of the pharmaceutical agent from the solid particle. The suspension can be prepared by any of the methods described herein. The active agent particles will have an average effective diameter of less than about 100 microns and the liquid phase contains non-lethal ingredients for the cells when added to a cell culture medium. The pharmaceutical composition may also include a surfactant, alone or in combination with other surfactants, to stabilize the pharmaceutical composition. The surfactant can be selected from a variety of anionic surfactants, cationic surfactants, zwitterionic surfactants, nonionic surfactants and known biological active surface modifiers.

The therapeutic agents can be selected from a variety of known pharmaceuticals, such as, but not limited to: analgesics, anesthetics, analeptics, adrenergic agents, adrenergic blocking agents, anticonvulsants, alkylating agents, alkaloids, allosteric inhibitors, anabolic steroids, anorexiantes , antacids, antidiarrheals, antidotes, antipyretics, antipyretics, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, anthelmintics, antibiotics, anticoagulants, antidepressants, antiepileptics, antifungals, antihistamines, antimuscarinic agents, antimicrobial agents, antineoplastic agents, agents antiprotozoals, antiviral agents, anxiolytic sedatives, beta-adrenoceptor blocking agents, contrast media, corticosteroids, cough suppressants, diagnostic agents, diagnostic imaging agents, dopaminergics, hemosta cosmetics, hematological agents, hypnotics, immunological agents, muscarinic agents, parasympathomimetics, prostaglandins, protease inhibitors, radiopharmaceuticals, sedatives, stimulants, sympathomimetics, vitamins, xanthines, growth factors, hormones and antipyretic agents. Antineoplastic agents may include paclitaxel and its derivative compounds, alkaloids, antimetabolites, enzyme inhibitors, alkylating agents and antibiotics. Other therapeutic agents include carbamazein, prednisolone and nabumetone. The therapeutic agents may also include a biological agent. The biological can be selected from proteins, polypeptides, carbohydrates, polynucleotides and nucleic acids. Nucleic acids include, but are not limited to, single or double stranded DNA, generally RNA and cDNA, t-RNA, mRNA, si-RNA and the like. The protein can be any antibody selected from polyclonal antibodies and monoclonal antibodies. Diagnostic agents include x-ray imaging agents and contrast media. Examples of x-ray imaging agents include WIN-8883 (ethyl 3,5-diacetamido-2,4,6-triiodobenzoate) also known as the diatrazoic acid ethyl ester (EEDA), WIN 67722, i.e. -ethoxy-6-oxohexyl-3,5-bis (acetamido) -2,4,6-triiodobenzoate; ethyl-2- (3,5-bis (acetamido) -2,4,6-triiodo-benzoyloxy) butyrate (WIN 1631 8), ethyl diatrizoxyacetate (WIN 12901), ethyl 2-3,5-bis (acetamido) -2,4,6-triiodobenzoyloxy) propionate (WIN 16923); N -ethyl 2- (3,5-bis (acetamido) -2,4,6-triiodobenzoyloxyacetamide (Wl 65312); isopropyl 2- (3,5-bis (acetamido) -2,4,6-triiodobenzoyloxy) acetamide (WIN 12855); diethyl 2- (3,5-bis (acetamido) -2,4,6-triiodobenzoyloxy malonate (WIN 67721); 2- (3,5-bis (acetamido) -2,4,6-triiodobenzoyloxy) ethyl phenylacetate (WIN 67585), propanodoic acid, [[3,5-bis (acetylamino) -2,4,5-triiodobenzoyl] oxy] bis (1-methyl) ester (WIN 68165), and benzoic acid , 3,5-bis (acetylamino) -2,4,5-triiodo-4- (ethyl-3-ethoxy-2-butenoate) ester (Wl 68209) Preferred contrast agents include those which are expected for relatively rapidly disintegrate under physiological conditions, thus minimizing any inflammatory response associated with a particle.Resintegration can result from enzymatic hydrolysis, solubilization of carboxylic acids at physiological pH, or other mechanisms.Thus, poorly soluble iodinated carboxylic acids, such as, yodipamide, diatriz acid oico and metrizoic acid, together with hydrolytically labile iodinated species, such as, WIN 67721, WIN 12901, WIN 68165 and WIN 68209 or others may be preferred. Other contrast media include, but are not limited to, particulate preparations of magnetic resonance imaging aids, such as gadolinium chelates, iron oxides, or other paramagnetic contrast agents. Examples of such compounds are gadopentetate dimeglumine (Magnevis®) and gadoteridol (Prohance®).

A description of classes of therapeutic agents and diagnostic agents and a list of species within each class can be found in Martindale, The Extra Pharmacopoeia, twenty-ninth edition, The Pharmaceutical Press, London, 1989, which is incorporated herein by reference and becomes a part hereof. The therapeutic agents and diagnostic agents are commercially available and / or can be prepared by techniques known in the art. Preferably, the pharmaceutical composition is a poorly water soluble compound. What is meant by "poorly soluble in water" is a solubility of the compound in water of less than about 10 mg / ml, and preferably less than 1 mg / ml. These poorly water soluble compounds are very suitable for aqueous suspension preparations, because they are limited alternatives for formulating these compounds in an aqueous medium. The following description of particles also applies to all embodiments of the present invention. The particles in the dispersion can be amorphous, semi-crystalline, crystalline, or a combination thereof, as determined by suitable analytical methods, such as differential scanning calorimetry (DSC) or X-ray diffraction. Prior to administration, the Pharmaceutical composition can be homogenized through a homogenization process. The pharmaceutical composition can also be homogenized through a microprecipitation / homogenization process, microprecipitation or ultrasonication as well. The dispersion of the pharmaceutical composition can be sterilized before being administered. The sterilization can be carried out by any medical sterilization process including heat sterilization or sterilization by gamma irradiation. It can also be sterilized by filtration, either directly as a dispersion having particle sizes below 200 nm, or by sterile filtration of the solutions used in the precipitation process, before forming the solid dispersion. Sterilization can also be achieved by brief application of very high pressure (more than 2000 atmospheres), or by a combination of high pressure and high temperature. The present invention can be practiced with water soluble compounds. These water-soluble active compounds are mixed with a polymer (eg, poly-polyglycollate copolymer (PLGA), polycyanoacrylate, albumin, starch), or are encapsulated in a surrounding vesicle which is impermeable to the pharmaceutical compound. This encapsulating vesicle may be a polymeric coating, such as, polyacrylate. In addition, the small particles prepared from these water-soluble compounds can be modified to improve the chemical stability and control the pharmacokinetic properties of the compounds by controlling the release of the compounds from the particles. Examples of water soluble compounds include, but are not limited to, simple organic compounds, proteins, peptides, nucleotides, oligonucleotides and carbohydrates. The particles used in the present invention have an average effective particle size of less than about 1 00 pm as measured by dynamic light scattering methods (e.g., photo-correlation spectroscopy, laser diffraction, low-angle laser light scattering). (LALLS), medium angle laser light scattering (MALLS)), light obscuration methods (Coulter method, for example), (HIAC) electrical resistance, rheology or microscopy (light or electrons). The preferred average effective particle size depends on factors, such as the intended route of administration, formulation, solubility, toxicity and bioavailability of the compound.

Preparation of the pharmaceutical composition as particles The processes for preparing the particles used in the present invention can be accomplished through numerous techniques known to those skilled in the art. A representative, but not exhaustive, discussion of techniques for preparing dispersions of particles of pharmaceutical compositions follows.

I. Energy addition techniques for forming small particle dispersions In general, the method for preparing small particle dispersions using energy addition techniques includes the step of adding the pharmaceutically active compound, which sometimes should be referred to as a medicament, in bulk form to a suitable vehicle, such as water or aqueous solution containing one or more of the surfactants set out below, or another liquid in which the pharmaceutical compound is not appreciably soluble, to form a first suspension, which it must be referred to as a presuspension. The energy is added to the presuspension to form a particle dispersion, which is more physically stable than the presuspension. Alternatively, the energy addition step can be applied to the dry powder form of the active compound. The energy is added by mechanical grinding (for example, bead milling, ball milling, hammer milling, fluid energy grinding, jet milling or wet grinding). Such techniques are described in U.S. Pat. 5, 145, 684, which is incorporated herein by reference and becomes a part of the present.

Energy addition techniques also include subjecting the presuspension to high cut conditions, including forces of cavitation, cutting or impact using a microfluidizer. The present invention further contemplates adding energy to the presuspension using a piston-opening homogenizer or counterflow current homogenizer, such as those described in U.S. Pat. 5,091, 188, which is incorporated herein by reference and becomes a part hereof. Piston opening homogenizers are commercially available under the product name EMULSIFLEX by Avestin, and French Pressure Cells sold by Spectronic Instruments. Suitable microfluidizers are available from Microfluidics Corp. The step of adding energy can also be accomplished using sonication techniques. The sonication step can be carried out with any suitable sonication device, such as Branson model S-450A or Cole-Parmer model 500/750 Watt. Such devices are well known in the industry. Normally, the sonication device has an oven or sonication probe that is inserted into the presuspension to emit the sonic energy in the solution. The sonication device, in a preferred form of the invention, is operated at a frequency from about 1 kHz to about 90 kHz and more preferably from about 20 kHz to about 40 kHz or any range or combination of ranges between them. The probe sizes may vary and are preferably of different sizes, such as 1 .27 cm (1/2 in) or 0.635 cm (1/4"n) or the like. Regardless of the energy addition technique used, the dispersion of small particles must be sterilized before use. Sterilization can be achieved by heat sterilization, gamma irradiation, filtration (either directly as a dispersion having particle sizes below 200 nm, or by sterile filtration of the solutions used in the precipitation process, before forming the solid dispersion), and by applying very high pressure (more than 2000 atmospheres), or by a combination of high pressure and high temperature.

I I. Precipitation methods for preparing particle dispersions with submicron dimensions Small particle dispersions can also be prepared by precipitation techniques. The following is a description of examples of precipitation techniques.

Microprecipitation Methods An example of a microprecipitation method is described in U.S. Pat. 5,780,062, which is incorporated herein by reference and becomes a part hereof. The '62 patent describes a process of organic compound precipitation that includes: (i) dissolving the organic compound in a first water-miscible solvent; (ii) preparing a polymer solution and an amphiphile in a second aqueous solvent and in said second solvent the organic compound is substantially insoluble, whereby a polymer / amphiphile complex is formed; and (iii) mixing the solutions of steps (i) and (ii), in order to cause precipitation of an aggregate of the organic compound and the polymer / amphiphile complex. Another example of a suitable precipitation process is described in U.S. Pat. 6,607,784 and US serials commonly ceded and co-pending us. 09 / 874,499; 09 / 874,637; and 1 0/021, 2, which are incorporated herein by reference and become a part of the present. The described processes include the steps of: (1) dissolving an organic compound in a first water miscible organic solvent to create a first solution; (2) mixing the first solution with a second solvent or water to precipitate the organic compound to create a presuspension; and (3) adding energy to the presuspension in the form of high cut or heat mixing to provide a dispersion of small particles. Optionally, the first organic solvent is removed from the mixture by any suitable means, such as centrifugation or filtration methods. Moreover, the continuous phase of the dispersion can be optionally replaced by any other continuous phase by removing the first continuous phase using methods such as centrifugation and filtration, adding a second continuous phase and subsequently redispersing the solid material in the second continuous phase. One or more optional surface modifiers discussed below may be added to the first organic solvent or the second aqueous solution.

Emulsion precipitation methods A suitable emulsion precipitation technique is described in the commonly assigned and co-pending US serial. 09 / 964,273, which is incorporated herein by reference and becomes a part hereof. In this approach, the process includes the steps of: (1) providing a multiphasic system having an organic phase and an aqueous phase, the organic phase having a pharmaceutically active compound therein; and (2) sonicate the system to evaporate a portion of the organic phase to cause precipitation of the compound in the aqueous phase to form a dispersion of small particles. The step of providing a multiphase system includes the steps of: (1) mixing a water-immiscible solvent with the pharmaceutically active compound to define an organic solution, (2) preparing a water-based solution with one or more active surface compounds , and (3) mixing the organic solution with the aqueous solution to form the multiphase system. The step of mixing the organic phase with the aqueous phase may include the use of piston-opening homogenizers, colloid mills, high-speed stirring equipment, extrusion equipment, manual vibration or vibration equipment, microfluidizer or other equipment or techniques for provide high cut conditions. The crude emulsion will have oil droplets in the water of a size of approximately less than 1 μ in diameter. The crude emulsion is sonicated to define a microemulsion and eventually to provide a dispersion of small particles. Another approach to preparing a dispersion of pea-sized particles is described in the American serial commonly assigned and co-pending no. 1 0/1 83.05, which is incorporated herein by reference and becomes a part hereof. The process includes the steps of: (1) providing a crude dispersion of a multiphase system having an organic phase and an aqueous phase, the organic phase having a pharmaceutical compound therein; (2) provide energy to the raw dispersion to form a fine dispersion; (3) freeze the fine dispersion; and (4) lyophilizing the fine dispersion to obtain small particles of the pharmaceutical compound. The small particles can be sterilized by the techniques discussed below or the small particles can be reconstituted in an aqueous and sterilized medium. The step of providing a multiphase system includes the steps of: (1) mixing a water-immiscible solvent with the pharmaceutically effective compound to define an organic solution; (2) preparing a water-based solution with one or more active surface compounds; and (3) mixing the organic solution with the aqueous solution to form the multiphase system. The step of mixing the organic phase and the aqueous phase includes the use of piston-opening homogenizers, colloidal mills, high-speed stirring equipment, extrusion equipment, manual agitation or vibration type, microfluidizer, or other eq. uipo or techniques to provide high cut conditions.

Solvent-antisolvent precipitation Small particle dispersions can also be prepared using the solvent-antisolvent precipitation technique described by Fessi et al. in U.S. Patent No. 5,118,528 and by Leclef et al. in U.S. Patent No. 5, 1 00,591, which are incorporated herein by reference and become a part hereof. Both processes include the steps of: (1) preparing a liquid phase of a biologically active substance in a solvent or a mixture of solvents to which one or more surfactants may be added; (2) preparing a second liquid phase of a non-solvent or a mixture of non-solvents, the non-solvent being miscible with the solvent or mixture of solvents for the substance; (3) add together the solutions of (1) and (2) with agitation; and (4) remove unwanted solvents to produce a dispersion of small particles. These methods are distinguished from those described under the previous section, "Microprecipitation Methods", because they do not provide a last step of adding energy to the suspension in the form of high cut or heat mixing.

Phase inversion precipitation Small particle dispersions can be formed using phase inversion precipitation as described in U.S. Pat. 6,235,224, 6, 143, 211 and U.S. patent application no. 2001/0042932, each of which is incorporated herein by reference and becomes a part hereof. Phase inversion is a term used to describe the physical phenomena by which a polymer dissolved in a continuous phase solvent system is inverted into a solid macromolecular network, in which the polymer is the continuous phase. One method to induce phase inversion is by adding a non-solvent to the continuous phase. The polymer undergoes a transition from a single phase to an unstable two phase mixture: polymer-rich and polymer-poor fractions. Micellar droplets of non-solvent in the polymer rich phase serve as nucleation sites and become polymer coated. The '224 patent discloses that phase inversion of polymer solutions under certain conditions and can cause spontaneous formation of discrete microparticles, including nanoparticles. The '224 patent describes dissolving or dispersing a polymer in a solvent. A pharmaceutical agent is also dissolved or dispersed in the solvent. For the step of planting crystals to be effective in this process, it is desirable that the agent be dissolved in the solvent. The polymer, the agent and the solvent together form a mixture having a continuous phase, wherein the solvent is the continuous phase. The mixture is then introduced in an excess of at least ten times of a non-miscible solvent to cause the spontaneous formation of the microencapsulated microparticles of the agent having an average particle size of between 10 nm and 10 pm. The particle size is influenced by the volume ratio of solvent: non-solvent, polymer concentration, the viscosity of the polymer-solvent solution, the molecular weight of the polymer and the characteristics of the solvent-non-solvent pair.

PH displacement precipitation Small particle dispersions can be formed by pH displacement precipitation techniques. Such techniques typically include a step of dissolving a medicament in a solution having a pH where the medicament is soluble, followed by the step of changing the pH to a point where the medicament is no longer soluble. The pH can be acidic or basic, depending on the particular pharmaceutical compound. The solution is then neutralized to form a dispersion of small particles. A suitable pH shift precipitation process is described in U.S. Pat. 5,665,331, which is incorporated herein by reference and becomes a part of the present. The process includes the step of dissolving the pharmaceutical agent together with a crystal growth modifier (CGM) in an alkaline solution and then neutralizing the solution with an acid in the presence of a surface active agent (s).surface modifiers, suitable, to form a dispersion of small particles of the pharmaceutical agent. The precipitation step can be followed by the diafiltration cleaning steps of the dispersion and then adjusting the concentration of the dispersion to a desired level. Other examples of pH displacement precipitation methods are described in U.S. Pat. 5,716,642; 5,662,883; 5,560,932; and 4,608,278, which are incorporated herein by reference and become a part hereof.

Infusion precipitation method. Infusion precipitation techniques suitable for forming small particle dispersions are described in US Pat. 4,997,454 and 4,826,689, which are incorporated herein by reference and become a part of the present. First, a suitable solid compound is dissolved in a suitable organic solvent to form a mixture of solvents. Then, a miscible non-solvent that precipitates with the organic solvent is infused into the solvent mixture at a temperature between about -1 0 ° C and about 1 00 ° C and at an infusion rate from about 0.01 ml per minute to about 1,000 per minute per volume of 50 ml to produce a suspension of precipitated non-aggregated solid particles of the compound with a substantially uniform average diameter of less than 10 μm. Agitation (for example, stirring) of the solution is preferred, being infused with the non-solvent that precipitates. The non-solvent may contain a surfactant to stabilize the particles against aggregation. The particles are then separated from the solvent. Depending on the solid compound and the desired particle size, the parameters of temperature, ratio of non-solvent to solvent, rate of infusion, rate of agitation and volume can be varied according to the invention. The particle size is proportional to the ratio of volumes of non-solvent: solvent and infusion temperature and is inversely proportional to infusion rate and agitation speed. The non-solvent that precipitates can be aqueous or non-aqueous, depending on the relative solubility of the compound and the desired suspension vehicle.

Temperature shift precipitation Temperature shift precipitation techniques can also be used to form small particle dispersions. This technique is described in U.S. Pat. 5, 188, 837, which is incorporated herein by reference and becomes a part of the present. In one embodiment of the invention, the lipospheres are prepared by the steps of: (1) melting or dissolving a substance, such as a medicament to be delivered in a molten vehicle to form a liquid of the substance to be delivered; (2) adding a phospholipid together with an aqueous medium to the molten substance or vehicle at a temperature higher than the melting temperature of the substance or carrier; (3) mixing the suspension at a temperature above the melting temperature of the vehicle until a homogeneous fine preparation is obtained; and then (4) rapidly cooling the preparation at room temperature or below.

Precipitation of solvent evaporation Precipitation techniques of solvent evaporation are described in the U.S. patent no. 4,973,465, which is incorporated herein by reference and becomes a part hereof. The '465 patent describes methods for preparing microcrystals including the steps of: (1) providing a solution of a pharmaceutical composition and a phospholipid dissolved in a common organic solvent or combination of solvents, (2) evaporating the solvent or solvents, and (3) ) suspending the film obtained by evaporating the solvent or solvents in an aqueous solution by vigorous stirring to form a dispersion of small particles. The solvent can be removed by evaporating a sufficient amount of the solvent to cause precipitation of the compound. The solvent can also be removed by any other known technique, such as applying a vacuum to the solution or blowing nitrogen over the solution.

Reaction Precipitation The reaction precipitation includes the steps of dissolving the pharmaceutical compound, and optionally other excipients, in a suitable solvent to form a solution. The compound can be added in an amount at or below the saturation point of the compound in the solvent. The compound or any of the excipients is precipitated from the solution by reacting with a chemical agent or by modification in response to adding energy, such as heat or UV light or the like, such that the modified compound has a lower solubility in the solvent and precipitates from the solution to form a small particle dispersion. The precipitation of excipient provides a solid matrix in which the drug is absorbed.

Precipitation of compressed fluid A suitable technique for precipitating by compressed fluid is described in WO 97/14407 for Johnston, which is incorporated herein by reference and is made a part hereof. The method includes the steps of dissolving a water-insoluble drug in a solvent to form a solution. The solution is then atomized into a compressed fluid, which can be a gas, liquid or supercritical fluid. The addition of the compressed fluid to a solution of a solute in a solvent causes the solute to reach or approach the supersaturated state and precipitate as fine particles. In this case, the compressed fluid acts as an anti-solvent, which lowers the cohesive energy density of the solvent in which the drug is dissolved. Alternatively, the medicament can be dissolved in the compressed fluid, which is then atomized into an aqueous phase. The rapid expansion of the compressed fluid reduces the solvent power of the fluid, which in turn causes the solute to precipitate as small particles in the aqueous phase. In this case, the compressed fluid acts as a solvent. In order to stabilize the particles against aggregation, a surface modifier, such as a surfactant, is included in this technique.

Atomization in cryogenic fluids A suitable technique for precipitating by compressed fluid is described by Williams et al. in the US application 10 / 273,730, which is incorporated herein by reference and becomes a part thereof. The method provides a system and method for the production of small particles, wherein the active ingredient is mixed with water, one or more solvents, or a combination thereof, and the resulting mixture is atomized at or below the surface of a cryogenic fluid. The frozen particles are provided by it. The materials for encapsulating the solid particles can also be added, so that the frozen particles are generated, wherein the encapsulating agent surrounds the active agent.

Protein microsphere precipitation The microspheres or microparticles used in this invention can also be produced from a process that involves mixing or dissolving macromolecules, such as proteins with a water-soluble polymer. This process is described in U.S. Patents 5,849,884, 5,981, 71 9, 6,090,925, 6,268,053, 6,458,387 and U.S. patent application no. 10 / 399,829, which are incorporated herein by reference and become a part hereof. In one embodiment of the invention, the microspheres are prepared by mixing a macromolecule in solution with a polymer or a mixture of polymers in solution at a pH close to the isoelectric point of the macromolecule. The mixture is incubated in the presence of an energy source, such as heat, radiation, or ionization, or alternatively, by removing energy, e.g., cooling, for a predetermined time. The resulting microspheres can be removed from any unincorporated component present in the solution by physical separation methods. There are several other methodologies for preparing dispersions of small particles. The present invention provides a methodology for terminally sterilizing such dispersions without significantly impacting the effectiveness of the preparation.

I I I. Additional Methods for Preparing Particulate Dispersions of Pharmaceutical Compositions The following additional processes for preparing particles of pharmaceutical compositions (ie, organic compound) used in the present invention can be separated into four general categories. Each of the process categories share the steps of: (1) dissolving an organic compound in a first solvent miscible with water to create a first solution, (2) mixing the first solution with a second solvent of water to precipitate the organic compound to create a pre-suspension, and (3) adding energy to the presuspension in the form of high cut or heat mixing, or a combination of both, to provide a stable form of the organic compound having the desired size ranges defined above. The mixing steps and the step of adding energy can be carried out in consecutive steps or simultaneously. Process categories are distinguished based on the physical properties of the organic compound as determined through x-ray diffraction studies, DSC studies, or another suitable study conducted before the energy addition step and after the addition step of energy. In the first process category, before the energy addition step, the organic compound in the presuspension takes an amorphous form, a semi-crystalline form or a supercooled liquid form and has an average effective particle size. After the energy addition step, the organic compound is in a crystalline form having an average particle size essentially equal to or less than that of the presuspension. In the second process category, before the addition step of enrgy, the organic compound is in a crystalline form and has an average effective particle size. After the energy addition step, the organic compound is in a crsitaline form having essentially the same average effective particle size as before the energy addition step, but the crystals after the energy addition step are less likely to be added or form large crystals. The lower tendency of the organic compound to aggregate or form large crystals is observed by dynamic laser light scattering and light microscopy. In the third process category, before the energy addition step, the organic compound is in a crystalline form that is friable and has an average effective particle size. What is meant by the term "friable" is that the particles are brittle and decompose more easily into smaller particles. After the energy addition step, the organic compound is in a crystalline form having an average effective particle size smaller than the crystals of the pre-suspension. By taking the steps necessary to place the organic compound in a crystalline form that is friable, the subsequent energy addition step can be performed more quickly and more efficiently when compared to an organic compound in a less friable crystalline morphology. In the fourth process category, the first solution and the second solvent are simultaneously subjected to the energy addition step. In this way, the physical properties of the organic copal before and after the energy addition step were not measured. The energy addition step can be performed in any manner where the presuspension or the first solution and the second solvent are exposed to cavitation, shear or impact forces. In a preferred form, the energy addition step is a tempering step. Tempering is defined in this invention as the process for converting matter that is thermodynamically unstable into a more stable form by simple or repeated application of energy (direct heat or mechanical stress), followed by thermal relaxation. This decrease in energy can be achieved by converting the solid form from a latex structure to a more orderly one. Alternatively, this stabilization can occur by rearrangement of the surfactant molecules at the solid-liquid interface. These four process categories are shown separately below. However, it should be understood that process conditions such as choice of surfactants or combination of surfactants, amount of surfactant used, reaction temperature, solution mixing speed, precipitation rate and the like, can be selected to allow any medication to be processed under any of the categories discussed below. The first process category, as well as the second, third and fourth process categories, can be further divided into two subcategories, Method A and B. The first solvent according to the following processes is a solvent or mixture of solvents, in which the organic compound of interest is relatively soluble and which is miscible in the second solvent. Such solvents include, but are not limited to, water-miscible protic compounds, in which a hydrogen atom in the molecule is attached to an electronegative atom, such as, oxygen, nitrogen, or other of Group VA, VIA, and VI IA in The periodic table. Examples of such solvents include, but are not limited to, alcohols, amines (primary or secondary), oximes, hydroxamic acids, carboxylic acids, sulfonic acids, phosphonic acids, phosphoric acids, amides and ureas. Other examples of the first solvent also include aprotic organic solvents. Some of these aprotic solvents can form hydrogen bonds with water, but they can only act as proton acceptors because they lack effective proton donor groups. One class of aprotic solvents is a dipolar aprotic solvent, as defined by the International Union of Pure and Applied Chemistry (IUPAC Compendium of Chemical Terminology, 2nd ed., 1 997): A solvent with a comparatively high relative permittivity (or dielectric constant) ), greater than ca. 15, and a dimensionable permanent dipole moment, which can not donate suitably labile hydrogen atoms to form strong hydrogen bonds, for example, dimethyl sulfoxide. The dipolar aprotic solvents can be selected from the group consisting of: amides (completely substituted, with nitrogen lacking bound hydrogen atoms), ureas (completely substituted, without hydrogen atoms attached to nitrogen) ethers, cyclic ethers, nitrites, ketones , sulfones, sulfoxides, completely substituted phosphates, phosphonate esters, phosphoramides, nitro compounds and similes. Dimethyl sulfoxide (DMSO), N-methyl-2-pyrrolidinone (NMP), 2-pyrrolidinone, 1,3-dimethylimidazolidinone (DMI), dimethylacetamide (DMA), dimethylformamide (DMF), dioxane, acetone, tetrahydrofuran (THF) , tetramethylene sulfone (sulfolane), acetonitrile and hexamethylphosphoramide (HMPA), nitromethane, among others, are members of this class. Solvents may also be chosen that are generally immiscible in water, but have sufficient solubility in water at low volumes (less than 10%) to act as a first solvent miscible in water at these reduced volumes. Examples include aromatic hydrocarbons, alkenes, alkanes and halogenated aromatics, halogenated alkenes and halogenated alkanes. Aromatics include, but are not limited to, benzene (substituted or unsubstituted), and monocyclic or polycyclic lows. Examples of substituted benzenes include, but are not limited to, xylenes (ortho, meta or para) and toluene. Examples of alkanes include, but are not limited to hexane, neopentane, heptane, isooctane and cyclohexane. Examples of halogenated aromatics include, but are restricted to, chlorobenzene, bromobenzene and chlorotoluene. Examples of halogenated alkanes and alkenes include, but are not restricted to, trichloroethane, methylene chloride, ethylene dichloride (EDC) and the like. Examples of all classes of prior solvents include, but are not limited to: N-methyl-2-pyrrolidinone (also called N-methyl-2-pyrrolidone), 2-pyrrolidinone (also called 2-pyrrolidone), 1, 3- dimethyl-2-imidazolidinone (DMI), dimethyl sulfoxide, dimethylacetamide, acetic acid, lactic acid, methanol, ethanol, isopropanol, 3-pentanol, n-propanol, benzyl alcohol, glycerol, butylene glycol (butanediol), ethylene glycol, propylene glycol, mono- and diacylated monoglycerides (such as glyceryl caprylate), dimethyl isosorbide, acetone, dimethylsulfone, dimethylformamide, 1,4-dioxane, tetramethylene sulfone (sulfolane), acetonitrile, nitromethane, tetramethylurea, hexamethylphosphoramide (HMPA), tetrahydrofuran (THF), dioxane, diethyl ether, tert-butylmethyl ether (TBME), aromatic hydrocarbons, alkenes, alkanes, halogenated aromatics, halogenated alkenes, halogenated alkanes, xylene, toluene, benzene, substituted benzene, ethyl acetate, methyl acetate, acetate of butyl, chlorobenzene, bromobenzene, chlorotoluene, trichloroethane, methylene chloride, ethylene dichloride (EDC), hexane, neopentane, heptane, isooctane, cyclohexane, polyethylene glycol (PEG, eg, PEG-4, PEG-8, PEG-9) , PEG-12, PEG-14, PEG-16, PEG-120, PEG-75, PEG-150), polyethylene glycol esters (examples such as PEG-4 dilaurate, PEG-2 dilaurate, PEG-6 isostearate , palm-stearate of PEG-8, palm-palmitostearate of PE G-150), polyethylene glycol sorbitan (such as PEG-20 sorbitan isostearate), polyethylene glycol monoalkyl ethers (examples such as PEG-3 dimethyl ether, PEG-4 dimethyl ether), polypropylene glycol (PPG), polypropylene alginate, PPG -10 butanediol, PPG-1 0 methyl glucose ether, PPG-20 methyl glucose ether, PPG-5 stearyl ether, propylene glycol dicaprylate / dicaprate, propylene glycol laurate and glycofurol (tetrahydrofurfuryl alcohol polyethylene glycol ether). A first preferred solvent is N-methyl-2-pyrrolidinone. Another preferred first solvent is lactic acid. The second solvent is an aqueous solvent. This aqueous solvent can be water by itself. This solvent may also contain buffers, salts, surfactant (s), water soluble polymers and combinations of these excipients.

Method A In Method A (see FIG.1), the organic compound ("medicine") is first dissolved in the first solvent to create a first solution. The organic compound can be added from about 0.1% (w / v) to about 50% (w / v) depending on the solubility of the organic compound in the first solvent. Heating the concentrate from about 30 ° C to about 100 ° C may be necessary to ensure complete dissolution of the compound in the first solvent. A second aqueous solvent is provided with one or more optional surface modifiers such as an anionic surfactant, a cationic surfactant, a zwitterionic surfactant, a nonionic surfactant or a molecule added thereto biologically active surface. Suitable anionic surfactants include, but are not limited to, alkyl sulfonates, alkyl phosphates, alkyl phosphonates, potassium laurate, triethanolamine stearate, sodium lauryl sulfate, sodium dodecylsulfate, polyoxyetne alkyl sulfates, sodium alginate, dioctyl sodium sulfosuccinate, phosphatidylglycerol, inosine phosphatidyl, phosphatidylinositol, diphosphatidylglycerol, phosphatidylserine, phosphatidic acid and their salts, carboximetilceulosa sodium, cholic acid and other bile acids (e.g., cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid, glicodeoxicólico acid) and salts the same (For example, sodium deoxycholate, etc.). Zwitterionic surfactants are electrically neutral but have local positive and negative charges within the same molecule. Suitable zwitterionic surfactants include, but are not limited to zwitterionic phospholipids. Suitable phospholipids include phosphatidylcholine, phosphatidylethanolamine, diacyl-glycero-phosphoethanolamine (such as dimyristoyl flicero-phosphoethanolamine (DMPE), dipalmitoyl-glycero-phosphoethanolamine (DPPE), distearoyl-glycero-phosphoethanolamine-(DSPE) and glycero-phosphoethanolamine dioleolil-( DOPE)). Phospholipid mixtures including anionic and zwitterionic phospholipids can be employed in this invention. Such mixtures include, but are not limited to lysophosphophoids, egg or soy phospholipid or any combination thereof. The phospholipid, whether anionic, zwitterionic or a mixture of phospholipids, can be salted or desalted, hydrogenated or partially hydrogenated or semi-synthetic natural or synthetic. The phospholipid may also be conjugated with a water soluble or hydrophilic polymer to specifically target delivery to macrophages in the present invention. However, conjugated phospholipids can be used to focus other cells or tissue in other applications. A preferred polymer is polyetne glycol (PEG), which is also known as monomethoxy polyetne glycol (mPEG). The molecular weights of the PEG can vary, for example, from 200 to 50,000. Some PEG's commonly used which are commercially available include PEG 350, PEG 550, PEG 750, PEG 1000, PEG 2000, PEG 3000 and PEG 5000. The phospholipid or the PEG-fosfolípdio may also incorporate a functional group which can bind covalent to a ligand include, but not limited to, proteins, peptides, carbohydrates, glycoproteins, antibodies or pharmaceutically active agents. These functional groups can be conjugated to the ligands through, for example, amide bond formation, disulfide or thioether formation, or biotin / streptavidin binding. Examples of functional groups of ligand binding include, but are not limited to, hexanoilamina, dodecanilamina, 1, 12-dodecanodicarboxilato, thioethanol, 4- (p-maleimidophenyl) butyramide (PB), 4- (p-maleimidometiI) cyclohexane- carboxamide (MCC), 3- (2-pyridyldithio) propionate (PDP), succinate, glutarate, dodecanoate and biotin. Suitable cationic surfactants include, but are not limited to, quaternary ammonium compounds, such as benzalkonium chloride, cetyltrimethylammonium bromide, chitosans, lauryldimethylbenzylamine chloride, acyl carnitine hydrochlorides, dimethyldioctadecylammonium bromide (DDAB), dioleoyltrimethylammonium (DOTAP), dimyristoyltrimethylammonium propane (DMTP), dimethylaminoethanecarbamoyl cholesterol (DC-Chol), 1,2-diacylglycerol-3- (0-alkyl) phosphocholine, O-alkyl phosphatidylcholine, alkyl pyridinium halides, or long chain alkyl amines, such as, for example, n-octylamine and oleylamine. Suitable nonionic surfactants include: glyceryl esters, polyoxyethylene fatty alcohol ethers (Macrogol and Brij), esters of polyoxyethylene sorbitan fatty acids (Polysorbates), esters of polyoxyethylene fatty acids (yrj), sorbitan esters (Span), glycerol monostearate, polyethylene glycols, polypropylene glycols, cetyl alcohol, cetostearyl alcohol, stearyl alcohol, aryl alkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxamers), poloxamines, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, non-crystalline cellulose, polysaccharides including starch and starch derivatives, such as hydroxyethyl starch (HES), polyvinyl alcohol and polyvinyl pyrrolidone. In a preferred form, the nonionic surfactant is a copolymer of polyoxyethylene and polyoxypropylene and preferably a block copolymer of propylene glycol and ethylene glycol. Such polymers are sold under the tradename POLOXAMER sometimes referred to as PLURONIC®, and sold by several suppliers including Spectrum Chemical and Ruger. Polyoxyethylene fatty acid esters include those having short alkyl chains. An example of such a surfactant is SOLUTOL® HS 15, polyethylene-660-hydroxystearate, manufactured by BASF Aktiengesellschaft. Biological active surface molecules include molecules such as albumin, casein, hirudin or other appropriate proteins. Polysaccharide biologics are also included, and consist of, but are not limited to, starches, heparins and chitosan. Other suitable surfactants include any amino acid, such as, leucine, alanine, valine, isoleucine, lysine, aspartic acid, glutamic acid, methionine, phenylalanine or any derivatives of these amino acids, such as, for example, amide or ester derivatives and polypeptides formed from these amino acids. It may also be desirable to add a pH adjusting agent to the second solvent. Suitable pH adjusting agents include, but are not limited to, hydrochloric acid, sulfuric acid, phosphoric acid, monocarboxylic acids (such as, for example, acetic acid and lactic acid), dicarboxylic acids (such as, for example, succinic acid ), tricarboxylic acids (such as, for example, citric acid), THAM (tri (hydroxymethyl) aminomethane), meglumine (N-methylglucosamine), sodium hydroxide, and amino acids, such as glycine, arginine, lysine, alanine, histidine and Leucine The second solvent should have a pH in the range from about 3 to about 1 1. The aqueous medium may additionally include an osmotic pressure adjusting agent, such as, but not limited to glycerin, a monosaccharide, such as dextrose, a disaccharide, such as sucrose, a trisaccharide, such as raffinose, and sugar alcohols, such as mannitol, xylitol and sorbitol. In a preferred form, the method for preparing small particles of an organic compound includes the steps of adding the first solution to the second solvent. The rate of addition is dependent on the size of the batch and the precipitation kinetics for the organic compound. Normally, for a small-scale laboratory process (1 liter preparation), the rate of addition is from about 0.05 ce per minute to about 10 ce per minute. During the addition, the solutions should be of constant agitation. It has been observed using luminous microscopy that amorphous particles are formed, semi-crystalline solids or a supercooled liquid to create a presuspension. The method further includes the step of subjecting the presuspension to an energy addition step to convert the amorphous particles, supercooled liquid or semicrystalline solid to a crystalline, more stable solid state. The resulting particles will have an average effective particle size as measured by dynamic light scattering methods. In the fourth process category, the first solution and the second solvent are combined while simultaneously conducting the energy addition step. The energy addition step involves adding energy through sonication, homogenization, countercurrent flow homogenization, microfluidization or other methods to provide impact, cut or cavitation forces. The sample can be cooled or heated during this stage. In a preferred form, the step of adding energy is effected by a piston-opening homogenizer, such as that sold by Avestin Inc. under the product designation EmulsiFlex-C160. In another preferred form, the energy addition step can be achieved by ultrasonication using an ultrasonic processor, such as the Vibra-Cell Utrasonic Processor (600 W), manufactured by Sonics and Materials, Inc. Still in another preferred form, the step of Energy addition can be achieved by the use of an emulsification apparatus as described in U.S. Pat. 5,720,551, which is incorporated herein by reference and becomes a part hereof. Depending on the rate of energy addition, it may be desirable to adjust the temperature of the processed sample to within the range of about -30 ° C to 30 ° C. Alternatively, in order to affect a desired phase change in the processed solid, it may also be necessary to heat the presuspension to a temperature within the range of about 30 ° C to about 100 ° C during the energy addition step.

Method B Method B differs from Method A in the following aspects. The first difference is that a surfactant or combination of surfactants is added to the first solution. The surfactants can be selected from the anionic, nonionic, cationic surfactant and active surface biological modifiers discussed above. A drug suspension resulting from the application of the processes can be administered directly as an injectable solution, provided that the Water for injection is used in the formulation and an appropriate medium for solution sterilization is applied. Sterilization can be achieved by methods well known in the art, such as steam or heat sterilization, gamma irradiation and the like. Other methods of sterilization, especially for particles in which more than 99% of the particles are less than 200 nm, would also include prefiltration first through a 3.0 micron filter, followed by filtration through a particle filter. 0.45 microns, followed by sterilization with heat or heat or sterile filtration through two membrane filters of 0.2 microns redundant. Yet another means of sterilization is sterile filtration of the concentrate prepared from the first solvent containing medicament and surfactant or optional surfactants and sterile filtration of the aqueous diluent. These are then combined in a sterile mixing vessel, preferably in a sterile, isolated environment. The mixing, homogenization and further processing of the suspension are carried out under aseptic conditions. Yet another method for sterilization would consist of heat sterilization or autoclaving within the homogenizer by itself, before, during or subsequent to the homogenization step. Processing after this heat treatment would be performed under aseptic conditions. Optionally, a solvent-free suspension can be produced by solvent removal after precipitation. This can be achieved by centrifugation, dialysis, diafiltration, force field fractionation, high pressure filtration, reverse osmosis, or other separation techniques well known in the art. The complete removal of N-methyl-2-pyrrolidione was normally carried out by one to three successive centrifugation runs; after each centrifugation (18,000 rpm for 30 minutes), the supernatant was decanted and discarded. A fresh volume of the suspension vehicle without the organic solvent was added to the remaining solids and the mixture was dispersed by homogenization. Those skilled in the art will recognize that other high cut mixing techniques could be applied in this reconstitution step. Alternatively, solvent-free particles can be formulated in various dosage forms as desired for a variety of routes of administration, such as oral, pulmonary, nasal, topical, intramuscular, and the like. Additionally, any unwanted excipient, such as surfactants, can be replaced by a more desirable excipient by using the separation methods described in the preceding paragraph.The solvent and first excipient can be discarded with the supernatant after centrifugation or A fresh volume of the suspension vehicle without the solvent and without the first excipient can then be added in. Alternatively, a new surfactant can be added, for example, a suspension consisting of medicament, N-methyl-2-pyrrolidinone ( solvent), poloxamer 188 (first excipient, sodium deoxycholate, glycerol and water) can be replaced with phospholipids (new surfactant), glycerol and water after centrifugation and removal of the supernatant.

I. First process category The methods of the first process category generally include the step of dissolving the organic compound in a water-miscible first solvent., followed by the step of mixing this solution with an aqueous solvent to form a presuspension, wherein the organic compound is in an amorphous form, a semicrystalline form or in a supercooled liquid form as determined by x-ray diffraction studies, DSC, light microscopy or other analytical techniques, and has an average effective particle size within one of the effective particle size ranges discussed above. The mixing step is followed by a step of energy addition.

I I. Second category of process The methods of the second category of process include essentially the same steps as in the steps of the first category of process but differ in the following aspect. A x-ray diffraction, DSC or other suitable anaytical technique of the presuspension shows the organic compound in a crystalline form and having an average effective particle size. The organic compound after the energy addition step has essentially the same average effective particle size as before the energy addition step, but has a lower tendency to aggregate into larger particles when compared to that of the presuspension particles. Without wishing to link to a theory, it is believed that the differences in particle stability may be due to a rearrangement of the surfactant molecules at the solid-liquid interface.

I I I. Third category of process The methods of the third category modify the first two steps of those of the first and second categories of processes to ensure that the organic compound in the presuspension is in a friable form having an average effective particle size (for example, such as slender needles and thin plates). Friable particles can be formed by selecting suitable solvents, surfactants or combination of surfactants, the temperature of the individual solutions, the mixing speed and precipitation rate and the like. The friability can also be enhanced by the introduction of latex defects (eg, cutting planes) during the mixing steps of the first solution with the aqueous solvent. This would arise by rapid crystallization, such as that provided in the precipitation step. In the energy addition step, these friable crystals are converted to crystals that are kinetically stabilized and have an average effective particle size smaller than those of the presuspension. Kinetically stabilized, it means particles that have a reduced tendency to aggregate when compared to particles that are not kinetically stabilized. In such a case, the step of adding energy results in a decomposition of the friable particles. By assuring that the particles of the presuspension are in a friable state, the organic compound can be prepared more easily and quickly in a particle within the desired size ranges, when compared to processing an organic compound where the steps have not been taken to do it in a friable way.

IV. Fourth process category The methods of the fourth process category include the steps of the first process category, except that the mixing step is performed simultaneously with the energy addition step.

Polymorphic Control The present process further provides additional steps to control the crystal structure of an organic compound to ultimately produce a suspension of the compound in the desired size range and a desired crystal structure. What is meant by the term "crystal structure" is the arrangement of the atoms within the unit cell of the crystal. It is said that compounds that can be crystallized in different crystal structures are polymorphic. The identification of polymorphs is an important step in the formulation of medication because different polymorphs of the same drug can show differences in solubility, therapeutic activity, bioavailability and suspension stability. Accordingly, it is important to control the polymorphic form of the compound to ensure product purity and reproducibility from batch to batch. Steps to control the polymorphic form of the compound include seeding the first solution, the second solvent or the presuspension to ensure the formation of the desired polymorph. Seeding includes using a seed compound or adding energy. In a preferred form, the seed compound is a pharmaceutically active compound in the desired polymorphic form. Alternatively, the seed compound can also be an inert impurity, a compound not related in structure to the desired polymorph, but with characteristics that can lead to quench the crystal core, or an organic compound with a structure similar to that of the polymorph wanted.

The seed compound can be precipitated from the first solution. This method includes the steps of adding the organic compound in sufficient quantity to exceed the solubility of the organic compound in the first solvent to create a supersaturated solution. The supersaturated solution is treated to precipitate the organic compound in the desired polymorphic form. Treating the supersaturated solution includes aging the solution for a period until the formation of a crystal or crystals is observed to create a seed mixture. It is also possible to add energy to the supersaturated solution to cause the organic compound to precipitate the solution in the desired polymorph. The energy can be added in a variety of ways including the energy addition steps described above. The additional energy can be added by heating, or by exposing the pre-suspension to electromagnetic energy sources, particle beam or electron beam. The electromagnetic energy includes luminous energy (ultraviolet, visible or infrared) or coherent radiation, so that provided by a laser, the microwave energy such as that provided by a maser (microwave amplification by stimulated emission of radiation), electromagnetic energy dynamic or other sources of radiation. It is also contemplated to use ultrasound, a static electric field or a static magnetic field, or combinations thereof, as the source of energy addition. In a preferred form, the method for producing seed crystals from an aged supersaturated solution includes the steps of: (i) adding an amount of an organic compound to the first organic solvent to create a supersaturated solution, (ii) aging the solution supersaturated to form detectable crystals to create a seed mixture; and (iii) mixing the seed mixture with the second solvent to precipitate the organic compound to create a presuspension. The presuspension can then be further processed as described in detail before, to provide an aqueous suspension of the organic compound in the desired polymorph and in the desired size range. The symmetry can also be achieved by adding energy to the first solution, the second solvent or the presuspension, provided that the liquid or exposed liquids contain the organic compound or a seed material. The energy can be added in the same way as described above for the supersaturated solution. Accordingly, the present processes utilize a composition of matter of an organic compound in a desired polymorphic form, essentially free of the polymorph or unspecified polymorphs. In a preferred form, the organic compound is a pharmaceutically active substance. It is contemplated that the methods described herein may be used to selectively produce a desired polymorph for numerous pharmaceutically active compounds.

B. Ex vivo Delivery of Solid Medicine Particulate There are numerous types of cells in the mammalian subject that are capable of phagocytosis and particle transport. These cells include, but are not limited to, macrophages, monocytes, granulocytes, neutrophils, basophils, eosinophils, and dendritic cells. The particles in the size range from about 150 nm to about 100 microns are more easily captured by these phagocytic organisms. Particles smaller than 150 nm may also associate in vitro and ex vivo with cells by binding to or association with the cell surface. Subsequently, they can be taken into cells by pinocytosis, which is an invagination of the cell membrane to form an intracellular capsule around the particle. In pinocytosis ("cell-drinking action"), the surrounding particle is relatively small (eg, 20 nm) (Watss, C; Marsh, M. Endocytosis: what goes in and how? (Endocytosis: what comes in and how?) J Cell Sci. 1992, 103 (1): 1 -8). Pinocytosis occurs continuously in almost all eukaryotic cells. The isolation of macrophages from the mammalian subject can be done by a cell separator. For example, the Isolex cell separator (Baxter Healthcare Corp., Deerfield, IL) can be used to isolate several cells. Other methods, known to those in the ex vivo cell isolation technique, could be employed to obtain cells useful in the methods of the present invention. Such methods include, but are not limited to, peripheral blood pheresis; by mobilization of bone marrow cells through G-CSF or GM-CSF, for example, or by direct removal of marrow cells by spinal, sternal, lumbar or iliac crest puncture.

Once isolated, the cells are nourished in a selective or non-selective growth medium and simultaneously or later they are contacted with the particulate pharmaceutical composition and incubated for a short period to allow the cellular uptake or adsorption of the particles. . The concentrations of pharmaceutical composition used in the ex vivo procedure will vary due to several factors, including, but not limited to, type of cells used, concentrations of cells, pharmaceutical agent used, size of small particle dispersions, disease to be treated, and so on. However, in general, cell isolates will be contacted with about 1 to about 300 mg / ml of a pharmaceutical composition of the present invention. The cells can be incubated with the pharmaceutical composition for up to 24 hours or more to allow sufficient cellular uptake of the drug particles. The uptake by the cells of the dispersion of the pharmaceutical composition as particles may include phagocytosis or other means of endocytosis, or adsorption of the particle on the surface of the cells. Additionally, in a preferred form of the invention, the particles during contact with the cells are at a concentration greater than the solidity of thermodynamic saturation, thereby allowing the particles to remain in particulate form during uptake and delivery to the mammalian subject. .

For marginally soluble drug, the ex vivo procedure can be used, provided that the isolated cells are able to surround (by endocytosis, phagocytosis, etc.), or adsorb the pharmaceutical composition particles at a faster rate than the competent dissolution process. The particles should be large enough to allow the cells to surround or adsorb the particles and deliver them to the desired target tissue prior to the complete dissolution of the particle. Additionally, the concentration of the pharmaceutical composition should be maintained higher than the saturation solubility of the composition, so that the particle is able to remain in the solid state during the round, adsorption or pinocytosis. Other cells can be used to deliver the pharmaceutically active compounds to a subject. Any cell type can be used in the present invention, as long as it is capable of capturing the active compound in the intracellular compartment of cultured cells, binding particles of the active compound to the periphery of such cells, or a combination of intracellular uptake and binding to the cell surface. Examples of other types of cells include: red blood cells, muscle cells, bone and bone marrow cells, vascular cells, organ tissue cells and neuronal cells. Those other cells can be isolated by techniques known to those skilled in the art. Any method for loading particles of pharmaceutically active compounds into cells can be used with the requirement that the method does not destroy or render otherwise useless the cells for administration to a subject. For example, the delivery of specific site of the particle via bio-recognition molecule can be used. See, for example, U.S. Patent Publication no. US 2003/0092069, incorporated herein by reference, which describes the delivery of specific site genes via a hollow nanoparticle. Other methods for loading the cells ex vivo include electroporation, sonoporation and other mechanical means that break the cell membrane (sonication, for example), and allow the insertion of solid particulate in the cells. Ultrasound was successfully used by Zarnisyn and Prausnitz (Zarnitsyn VG, Prausnitz MR, Physical parameters, nfluencing optimization of ultrasound-mediated DNA transfection.) (Physical parameters that influence the optimization of ultrasound-mediated DNA transfection.) Ultrasound Med Biol.; 30 (4): 527-38) to transiently break cell membranes and thereby facilitate loading of DNA into viable cells. Other mechanical methods are well known to those skilled in the art and are included as part of this description. Chemical methods to transiently destabilize cell membranes are also well known. Transfection reagents contain surface active and include 293fectin ™ Transfection Reagent and Lpofectamine ™, both products from Invitrogen Corporation (Carlsbad, California). Another example of a surfactant used to transfer DNA into cells is the SAINTMR reagent from Synvolux Therapeutics B.V. L.J. (Groningen, The Netherlands), which is based on a pyridinium surfactant. The ex vivo cells are nourished in a cell culture medium or other isolating system known to those skilled in the art. Examples of such media are Alserver solution, Ames medium, Eagle's basal medium, CHO cell culture medium (Chinese hamster ovary), Click medium, Dulbecco's modified Eagle medium, phosphate buffered saline, dextrose or sucrose. phosphate-buffered, Earle's balanced salt solution, gene-3 therapy medium, Gey's balanced salt solution, Glasgow's minimal essential medium, Hanks balanced salt solutions, hybridoma medium, Iscove's modified Dulbecco's medium, buffer of Krebs-Henseleit with sugars, Leibovitz medium (L-15), medium M16, medium of McCoy, MCDB, MDBK (bovine kidney of Madin-Darby), MDCK (canine kidney of Madin-Darby), medium 199, NCTC, Ham media (e.g., Nutrient Mixture F-10), Coon modified Ham medium, RPMI, and others, such as those listed in Biochemicals & Reagents for Life Science Research, Sigma-Aldrich Co. (St. Louis, MO, US). The purpose of the culture thus described may be for the purpose of simple storage without cell loss, or for cell expansion, by appropriate addition of growth factors, cytokines and nutrients, to encourage cell expansion. Such expansion would minimize the number of times a patient would have to be prepared for removal of cell samples. The loaded cells can be administered intravenously, intramuscularly, subcutaneously, intradermally, intra-articularly, intrathecally, epidurally, intracerebrally, by buccal, rectal, topical, transdermal, oral, intranasal administration, by pulmonary route, intraperitoneal, intra-ophthalmic, retro-orbital, or through any procedure that can be used for delivery of cells loaded with a particle to the mammalian subject. The administration step can be by bolus injection, by intermittent infusion or by continuous infusion. The amount of cells and method of delivery will be determined by expert clinicians. Several factors will affect cell concentration and delivery method including, but not limited to, the type of cells used, the sex, weight and age of the subject to be treated, the type and maturity of the disease or disorder to be treated, the agent Pharmaceutical charged in the cells and so on. Certain viruses and bacteria can be picked up by phagocytic cells and continue to remain inside these cells. However, cells loaded with the drug particles are effective in treating such infections because the drug is concentrated in the phagocytic cells and the infected organism is exposed to much larger amounts of the drug, thereby killing the organism. Additionally, after perfusing in infected tissues, the solubilizable particles in acid dissolve due to lower pH levels within the phagocytic cells, thereby releasing the drug concentrations. A concentration gradient is formed with higher concentrations of the pharmaceutical composition within an endosomal body of phagocytic cells and lower concentrations outside the endosome. In this way, the contents of the particles within the macrophage are released into the surrounding tissue for relief purposes. Over time, free viral and bacterial organisms that reside in the surrounding tissue are exposed to the drug at higher concentrations than that which is normally capable of being delivered through the administration of the free drug, not encapsulated as such. Although specific modalities have been illustrated and described, numerous modifications come to mind without departing from the spirit of the invention and the scope of protection is limited only by the scope of the accompanying claims.

Claims (1)

1. CLAIMS 1 . A method for the preparation and delivery of small particles of pharmaceutically active agents to a mammalian subject comprising: (i) collecting tissue cells from a mammalian donor, (ii) nourishing the cells in a cell culture medium to which it is added a pharmaceutical composition comprising the particles of one or more therapeutically active compounds or diagnostic agents, (iii) causing the particles to be picked up by the cells in either an intracellular compartment of the cells, binding of the particles to a periphery of the cells, or a combination thereof; and (iv) administering the cells to a mammalian subject. The method of claim 1, wherein the step of administering the cells comprises the step of delivering the cells to an objective tissue of the mammalian subject. The method of claim 1, wherein the step of administering comprises the step of administering the cells intravenously, intramuscularly, subcutaneously, intradermally, intra-articularly, intrathecally, epidurally, intracerebrally, buccally, rectally, topically, transdermally, oral, intranasal, via the pulmonary route, intraperitoneal, intra-ophthalmic or combinations thereof. The method of claim 1, wherein the cells are capable of phagocytosis, adsorption of the pharmaceutical composition as particles, or pinocytosis of these particles. The method of claim 1, wherein the cells are selected from the group consisting of macrophages, monocytes, granulocytes, neutrophils, basophils, eosinophils, dendritic cells and combinations thereof. The method of claim 1, wherein the cells are selected from the group consisting of red blood cells, muscle cells, bone and bone marrow cells, vascular cells, organ tissue cells, neuronal cells, dendritic cells and combinations of the same. The method of claim 1, wherein the step of nourishing the cells comprises the step of isolating the cells. The method of claim 7, wherein the step of isolating the cells is performed by a cell separator or apheresis device. The method of claim 2, wherein a portion of the particles do not dissolve prior to delivery to the target tissue. The method of claim 1, wherein the composition has a concentration of particles above the saturation solubility of the compound (s). eleven . The method of claim 1, wherein the compound or compounds are poorly soluble in water. The method of claim 1, wherein the step of nourishing cells is performed over a period of up to 24 hours. The method of claim 1, wherein the pharmaceutical composition further comprises a surfactant. 14. The method of claim 13, wherein the surfactant is selected from the group consisting of anionic surfactants, cationic surfactants, zwitterionic surfactants, nonionic surfactants, active surface biological modifiers and combinations thereof. The method of claim 1, wherein the particles in the composition are amorphous, semi-crystalline or a combination thereof, as determined by either differential scanning calorimetry or X-ray diffraction. 16. The method of the claim 1, wherein the pharmaceutical composition is miscible in water. The method of claim 12, wherein the therapeutic agent is selected from the group consisting of: analgesics, anesthetics, analeptics, adrenergic agents, adrenergic blocking agents, anticonvulsants, alkylating agents, alkaloids, allosteric inhibitors, anabolic steroids, anorexiaters, antacids, antidiarrheals, antidotes, antipyretics, antipyretics, antirheumatic agents, psychotherapeutic agents, neural blocking agents, anti-inflammatory agents, anthelmintics, antibiotics, Anticoagulants, antidepressants, antiepileptics, antifungals, antihistamines, antimuscarinic agents, antimycobacterial agents, antineoplastic agents, antiprotozoal agents, antiviral agents, anxiolytic sedatives, blocking agents beta-adrenoceptor, contrast media, corticosteroids, cough suppressants, diagnostic agents, agents diagnostic imaging, dopaminergics, haemostatics, hematological, hypnotics, inmuriológicos agents, muscarinic, parasympathomimetics, prostaglandins, protease inhibitors, radio-pharmaceuticals, sedatives, stimulants, sympathomimetics, vitamins, xanthines, factors crecmiento, hormones, antipriones agents and combinations thereof. The method of claim 17, wherein the antineoplastic agent is selected from the group consisting of: paclitaxel and its derivative compounds, alkaloids, antimetabolites, enzyme inhibitors, alkylating agents, antibiotics, gene therapy agents, and combinations thereof . The method of claim 12, wherein the therapeutic agent is selected from the group consisting of oligonucleotides and nucleic acids. The method of claim 12, wherein the therapeutic agent is a biological agent. twenty-one . The method of claim 20, wherein the biological is selected from the group consisting of proteins, polypeptides, carbohydrates, polynucleotides, nucleic acids and combinations thereof. 22. The method of claim 21, wherein the protein is an antibody selected from the group consisting of polyclonal antibodies, monoclonal antibodies and combinations thereof. 23. The method of claim 1, wherein the dispersion of the pharmaceutical composition is sterilized prior to administration. The method of claim 1, wherein the therapeutic active compound is used to treat genetically acquired or hereditary diseases. 25. The method of claim 24 wherein the genetically acquired or inherited disease is selected from the group consisting of sickle cell disease, Burkitt lymphoma, Gaucher's disease, hemophilia, chronic myeloid leukemia, Niemann-Pick disease, paroxysmal nocturnal hemoglobinuria, porphyria, thalassemia, breast and ovary, colon cancer, carcinoma small cell lung, malignant melanoma, multiple endocrine neoplasia, neurofibromatosis, pancreatic cancer, polycystic kidney disease, prostate cancer, retinoblastoma, tuberous sclerosis , Von Hippel-Lindau disease, colon cancer, Crohn's disease, cystic fibrosis, type 1 diabetes, malabsorption alactosa glucose, Wilson's disease, Zellweger syndrome, genetically acquired deafness, neurofibromatosis, Pendred syndrome, Best's disease, genetically acquired glaucoma, sinusoidal atrophy of the choroid and retina, retinoblastoma, s ome Rett, adrenal hyperplasia, congenital adrenoleukodystrophy, polyglandular autoimmune syndrome, Cockayne syndrome, diastrophic dysplasia, multiple endocrine neoplasia, ataxia telangiectasia, atherosclerosis, long QT syndrome, Williams syndrome, asthma, ataxia telangiectasia, DiGeorge syndrome, immunodeficiency with hyper-lgM, severe combined immunodeficiency, Alport syndrome, male pattern baldness, prostate cancer, Fanconi anemia, Hartnup's disease, Kartagener's syndrome, lysosomal storage diseases and pyruvate dehydrogenase deficiency. 26. A composition for delivery to a mammalian subject comprising a dispersion of a pharmaceutical composition provided as particles having an average particle size of less than about 100 microns and adapted to be administered to the mammalian subject for delivery to tissues of an effective amount of the composition pharmaceutical by means of cells capable of reaching tissues. The composition of claim 26, wherein the cells are capable of phagocytosis, adsorption of the pharmaceutical composition as particles on cell surfaces, pinocytosis or combinations thereof. The composition of claim 26, wherein the cells are selected from the group consisting of macrophages, monocytes, granulocytes, neutrophils, basophils, eosinophils, base cells, dendritic cells and combinations thereof. 29. The composition of claim 26, wherein the cells are selected from the group consisting of red blood cells, muscle cells, bone marrow and bone marrow cells, vascular cells, organ tissue cells, neuronal cells and combinations thereof. 30. The composition of claim 26, wherein the pharmaceutical composition is combined with the cells in a selective or non-selective culture medium. 31 The composition of claim 26, wherein the therapeutic agent is a biological agent. 32. The composition of claim 31, wherein the biological is selected from the group consisting of proteins, polypeptides, carbohydrates, polynucleotides, nucleic acids and combinations thereof. 33. The composition of claim 32, wherein the protein is an antibody selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and combinations thereof. 34. The composition of claim 26, wherein the dispersion of the pharmaceutical composition is delivered intravenously, intramuscularly, subcutaneously, intradermally, intra-articularly, intrathecally, epidurally, intracerebrally, via buccal route, rectal, topical, transdermal, oral, intranasal , via pulmonary route, intraperitoneal, intra-ophthalmic, retro-orbital or combinations thereof. 35. The composition of claim 26, wherein the dispersion of the pharmaceutical composition is sterilized before administration. SUMMARY The present invention concerns a method for preparing and delivering small particles of a pharmaceutically active material to a mammalian subject for treating diseases or disorders. A preferred embodiment comprises: (i) harvesting tissue cells from an animal donor, (ii) selective or non-selective growth of these cells in a cell culture medium to which solid particles of a therapeutically active compound are added, in their free majority of a drug carrier (approximately 10% or less, by weight), and having an average particle size of less than about 100 microns, (iii) contacting the cells in the cell culture medium with the solid particles of compound Therapeutically active, which causes the particles to be picked up by the cells either in the intracellular compartment of the cultured cells, binding of the active compound as particles to the periphery of scells, or a combination of intracellular uptake and cell surface binding ( V) optionally, isolation and / or resuspension of the cells prepared in steps iii, (v) administering the cells to the suj eto mammal. The pharmaceutically active material can be administered intravenously, intramuscularly, subcutaneously, intradermally, intra-articularly, intrathecally, epidurally, intracerebrally, via the buccal route, rectally, topically, transdermally, orally, intranasally, via the pulmonary route, intraperitoneally or combinations of the same. After administration, the loaded cells transport the pharmaceutical composition as particles.